The present invention relates to a composition for modifying the mobility of DNA in a sieving or non-sieving matrix, and to methods of using the composition for determining the sequence of a target polynucleotide.
Miniaturized electrophoresis systems have the potential to substantially increase the speed and throughput of automated DNA sequencing while reducing the overall cost per base. Current miniaturized electrophoresis systems, in particular capillary electrophoresis, continue to rely upon the use of viscous polymer solutions as xe2x80x9cgelsxe2x80x9d that provide a physical separation of DNA fragments according to chain length.
Unfortunately, gel-based methods for DNA sequencing have a maximum read lengths of about 1000 bases, with a typical read length being at most about 550 to about 650 bases. Since engineered DNA polymerases can produce more than 2500 bases of sequence per Sanger sequencing reaction, even optimized sequencing gels essentially xe2x80x9chidexe2x80x9d or xe2x80x9closexe2x80x9d about 1500 bases of information per reaction. Moreover, the replacement of viscous polymer gels from chip microchannels is difficult, if not impossible, thus preventing the use of automated chip-based gel electrophoresis systems for multiple, consecutive DNA sequencing analysis.
In addition to the above-described read-length limitations of gel electrophoresis, there is another drawback to the use of gels that makes them particularly inconvenient for capillary and microchip geometries. A reusable sequencing chip or capillary array must allow gels to be replaced before each run to eliminate sample carry-over and avoid irreproducibilities and current failures that may result from chemical- and/or field-induced gel breakdown. Unfortunately, no replaceable, high-performance sequencing gel appropriate for the microchannels on CE chips is currently available. And since sequencing gels cannot be replaced from chip microchannels, each chip is essentially a single-use device, an outcome that will greatly increase associated operating costs and decrease the convenience of miniaturized DNA sequencers.
Moreover, the sequencing gels used in experimental microchips currently are typically polymerized in situ, which typically yields low-performance gels because acrylamides generally do not polymerize to high molecular weights or yield greater than 85% conversion, i.e., polymerization, in a confined microchannel. While extremely xe2x80x9clargexe2x80x9d channels may be fabricated onto the chip (e.g., 100 xcexcm widexc3x9790 xcexcm deep), which allows viscous gels to be forced into the channels under pressure, these large channels, however, result in broadened DNA bands and/or reduced separation efficiency.
Therefore, there is a need for a method for electrophoretic DNA sequencing that does not demand the use of high-viscosity physical gels or cross-linked chemical gels. There is also a need for novel sequencing technologies that will substantially increase the speed and throughput of automated sequencing instruments while reducing the overall cost per base.
The present invention provides a compound and methods for using the same in electrophoretic separation of binding polymers. In particular, the present invention provides a polyamide compound comprising at least one hydrophilic C1-C10 hydrocarbyl substituent on an amide nitrogen atom and methods for using the polyamide in electrophoretic separation of binding polymers in a non-sieving liquid medium.
One aspect of present invention provides a polyamide of the formula: 
where
L1 is selected from the group consisting of H, amide protecting groups and moieties of the formula: 
each X is independently an amino acid side-chain residue or of the formula xe2x80x94CH2SL2 or xe2x80x94(CH2)3NL3R4;
each L2 is independently a thiol protecting group or a moiety of the formula: 
each L3 is independently H, an amine protecting group, an xcex1, xcex2-unsaturated carbonyl moiety or a conjugate moiety of the formula: 
provided at most one and only one L3 is the conjugate moiety;
each L4 is independently H, amide protecting groups or the moiety of the formula: 
each R is independently xe2x80x94CH2xe2x80x94 or 
each R1 is independently H, a protecting group or C1-C10 hydrocarbyl;
each of R2 and R3 are independently H, C1-C6 alkyl, or an amide protecting group;
each R4 is independently H, an amine protecting group, or C1-C6 alkyl, provided at least one of L3 or R4 on the same nitrogen atom is not H;
each R5 is independently C1-C10 alkylene;
aaal is the polynucleotide moiety;
p1 is H, C1-C6 alkyl or an amine protecting group;
a is an integer from 1 to 200;
each b is independently an integer from 1 to 200;
c is an integer from 1 to 10;
d is an integer from 1 to 50; and
each e is independently an integer from 1 to 200.
Preferably, for polyamide of formula I, at least one R1 is hydrophilic C1-C10 hydrocarbyl.
Another aspect of the present invention provides a polyamide of the formula: 
where
each R10 is independently H or a carboxylic acid protecting group;
each R11 is independently H, a protecting group or C1-C10 hydrocarbyl;
q is an integer from 1 to 1,200; and
each Q1 is independently an amino acid side-chain residue or a derivative thereof, provided at least one Q1 is an amino acid side-chain residue derivative of the formula: 
each of the moiety xe2x80x94Q3xe2x80x94X1xe2x80x94is an amino acid side-chain residue having xe2x80x94X1H functional group;
each X1 is independently O, S or NP2;
L is a linker comprising C1-C6 alkylene with carbonyl groups on both of the terminal groups; and
each of L1, L2, L3, L4, P1, R, R1, R2, R3, R4, R5, X, a, b, c, d, and e is independently those described above.
Another embodiment of the present invention provides a polyamide-polynucleotide primer conjugate and method for determining the nucleotide sequence of a target nucleic acid which comprises the steps of:
(a) annealing a polyamide-polynucleotide primer conjugate to the target nucleic acid, wherein the polyamide moiety comprises at least one hydrophilic C1-C10 hydrocarbyl substituent on an amide nitrogen atom;
(b) extending the primer with a nucleic acid polymerase in the presence of nucleoside triphosphate precursors and at least one chain terminating nucleotide, thereby forming conjugated nucleic acid fragments;
(c) separating the conjugated nucleic acid fragments by electrophoresis in a non-sieving matrix; and
(d) determining the nucleotide sequence of the target nucleic acid by the separated nucleic acid fragments.
Preferably, the polyamide-polynucleotide primer conjugate comprises a thioether linkage between a polyamide moiety and a polynucleotide moiety. Preferably, the polyamide-polynucleotide primer conjugate is a polypeptoid-polynucleotide primer conjugate of formula I or polypeptide-polynucleotide primer conjugate of formula II above comprising at least one L3 where one and only one L3 is the conjugate moiety of the formula: 
where aaal is the polynucleotide moiety, wherein the hydroxy group of the terminal 5xe2x80x2-position of the polynucleotide moiety has been replaced with a thiol group to form the thioether linkage between the polyamide moiety and the polynucleotide moiety.
Another embodiment of the present invention provides a method for producing a polyamide of the formula: 
comprising contacting a nucleophilic compound of the formula: 
with an xcex1, xcex2-unsaturated carbonyl of the formula: 
under conditions sufficient to produce the polyamide III, where
L5 is a moiety of the formula: 
L6 is a moiety of the formula: 
Y1 is S or NP2;
each P2 is independently H, C1-C6 alkyl or an amine protecting group;
each R13 is C1-C6 alkylene;
m is an integer from 1 to 200;
n is an integer from 1 to 200;
x is an integer from 0 to 200;
y is an integer from 0 to 10;
z is an integer from 0 to 50;
s is an integer from 0 to 200;
t is an integer from 0 to 10;
u is an integer from 0 to 50; and
each of L2, L3, L4, P1, R, R1, R2, R3, R4, X, and e is independently those described above.
Still another embodiment of the present invention provides a method for producing a polyamide of the formula: 
comprising contacting a polypeptide of the formula: 
with a polypeptide of the formula: 
under conditions sufficient to produce polyamide VI, where
L7 is a moiety of the formula xe2x80x94C(xe2x95x90O)xe2x80x94R12xe2x80x94C(xe2x95x90O)OM;
M is H or a metal;
each q1 is independently an integer from 0 to about 40;
each q2 is independently an integer from 1 to about 40;
each q3 is independently an integer from 0 to about 40;
q4 is an integer from 1 to about 50;
L is as described above, preferably a moiety of the formula xe2x80x94C(xe2x95x90O)xe2x80x94R12xe2x80x94C(xe2x95x90O)xe2x80x94;
R12 is C1-C6 alkylene; and
each of L1, L2, L3, L4, P1, Q1, the moiety xe2x80x94Q3xe2x80x94X1xe2x80x94, R, R1, R2, R3, R4, R5, R10, R11, X, X1, a, b, c, d, and e is independently those described above.
Still yet another embodiment of the present invention provides a method for forming a bond between a fused silica surface and a polyamide comprising:
(a) contacting the surface of said fused silica with an oxidizing agent to form an oxidized surface comprising a hydroxy group;
(b) silanizing the oxidized surface of the fused silica with a silanizing agent to form a silanized surface;
(c) contacting the silanized surface with a linking reagent to form a surface having silanized linker; and
(d) contacting the silanized linker with the polyamide to form a covalent bond between the silanized linker and the polyamide.
In one particular aspect of bond formation between the fused silica surface and the polyamide, the polyamide is a polypeptoid of the formula: 
which forms the covalent bond between the fused silica and the functional group X, where
each of P1, R1, R2 and R3 is independently those described above;
X is as described above, preferably xe2x80x94CH2SH or xe2x80x94(CH2)3NHR2; and
w is an integer from 1 to 200.
In another aspect of bond formation between the fused silica surface and the polyamide, the polyamide is a polypeptide of formula II above provided the polypeptide comprises at least one X which forms the covalent bond between the fused silica and the functional group X.